H-Y (male-specific) antigen appears to play important roles in gonadal organogenesis in reproduction and the materno-fetal relationship, and in clinical transplantation. Detailed study of these functions has been hindered by the lack of a simple method for the detection of H-Y antigen and its localization as a cellular or subcellular level. Furthermore, H-Y antigen has never been purified and consequently all currently available antisera are of necessity prepared in inbred strains of mice. The objectives of this project are thus to obtain preliminary physico-chemical information concerning H-Y antigen so that purification can be achieved from a suitable human tissue source, and antisera raised using the purified material. The project may be considered conveniently in three phases. Firstly, a variety of male tissues and male-derived cell lines will be screened for the presence of H-Y antigen using our fluorescence microscopy technique with appropriately absorbed mouse H-Y antisera. Secondly, homogenates will be made of selected tissues and purification of H-Y antigen will be attempted using a combination of isoelectric focusing and affinity chromatography. Following immunological recognition of the bands representing H-Y antigen in analytical gels, larger scale recovery of protein will be attempted using preparative isoelectric focusing in granular gels. The yeild of protein can also be increased by the use of affinity chromatography. A direct technique involving coupling of H-Y antisera to agarose gels will be carried out. However, since denaturation of bound protein may occur upon its removal with chaotropic agents, a subtractive technique will be employed in which antisera to homogenates of female tissues are coupled to agarose gels. Thirdly, the resultant purified preparation of H-Y antigen will be used to immunize virgin female rabbits. The antisera obtained will be absorbed as appropriate with female tissues to remove contaminating specificities. Such antisera should contain H-Y antbodies at high titre.